Hydrogen peroxide induces apoptosis-like death in Leishmania donovani promastigotes

Abstract

Leishmania donovani promastigotes introduced into the bloodstream by sandfly vectors, are exposed to reactive oxygen species like H₂O₂ during phagocytosis by the host macrophages. H₂O₂ can induce promastigote death, but the mechanism of induction of this death is not known. Studies presented in this paper demonstrate that exposure to 4 mM H₂O₂ results in a pattern of promastigote death that shares many features with metazoan apoptosis. Motility and cell survival in these parasites show a gradual decline with increasing doses of H₂O₂. Features common to metazoan apoptosis, such as nuclear condensation, DNA fragmentation with accompanying DNA ladder formation and loss of cell volume, are observed after exposure to 4 mM H₂O₂. Within 30 minutes of the exposure, there is a significant increase in the ability of the cell lysates to cleave the fluorogenic tetrapeptide acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin, which is a substrate for the CED-3/CPP32 group of proteases. Pretreatment of cells with a specific inhibitor of CED-3/CPP32 group of proteases, Z-DEVD-FMK, reduces the number of cells showing apoptosis-like features, prevents DNA breakage and inhibits cleavage of a PARP-like protein. Activation of the caspase-like proteases is followed at 2 hours by the cleavage of a poly(ADP)ribose-polymerase-like protein and a reduction in intracellular glutathione concentration. DNA breakdown as detected by TdT labelling of cells and agarose gel electrophoresis is visible at 6 hours. Taken together, the above data show for the first time that there is a distinct pathway for apoptosis-like death in L. donovani.